Studies on Activities and Chemical Characterization of Medicinal Plants in Search for New Antimalarials: A Ten Year Review on Ethnopharmacology

Malaria is an endemic disease that affected 229 million people and caused 409 thousand deaths, in 2019. Disease control is based on early diagnosis and specific treatment with antimalarial drugs since no effective vaccines are commercially available to prevent the disease. Drug chemotherapy has a strong historical link to the use of traditional plant infusions and other natural products in various cultures. The research based on such knowledge has yielded two drugs in medicine: the alkaloid quinine from Cinchona species, native in the Amazon highland rain forest in South America, and artemisinin from Artemisia annua, a species from the millenary Chinese medicine. The artemisinin-based combination therapies (ACTs), proven to be highly effective against malaria parasites, and considered as “the last bullet to fight drug-resistant malaria parasites,” have limited use now due to the emergence of multidrug resistance. In addition, the limited number of therapeutic options makes urgent the development of new antimalarial drugs. This review focuses on the antimalarial activities of 90 plant species obtained from a search using Pubmed database with keywords “antimalarials,” “plants” and “natural products.” We selected only papers published in the last 10 years (2011–2020), with a further analysis of those which were tested experimentally in malaria infected mice. Most plant species studied were from the African continent, followed by Asia and South America; their antimalarial activities were evaluated against asexual blood parasites, and only one species was evaluated for transmission blocking activity. Only a few compounds isolated from these plants were active and had their mechanisms of action delineated, thereby limiting the contribution of these medicinal plants as sources of novel antimalarial pharmacophores, which are highly necessary for the development of effective drugs. Nevertheless, the search for bioactive compounds remains as a promising strategy for the development of new antimalarials and the validation of traditional treatments against malaria. One species native in South America, Ampelozyzyphus amazonicus, and is largely used against human malaria in Brazil has a prophylactic effect, interfering with the viability of sporozoites in in vitro and in vivo experiments.


INTRODUCTION
Human malaria is an infectious disease caused by single-cell protozoan parasites of the Plasmodium genus, namely: P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, and is transmitted through the bite of female Anopheles mosquitoes (Crompton et al., 2014;Singh et al., 2021); of these, P. falciparum is the most virulent and prevalent globally. Symptoms can range from a mild to severe disease which can cause important physical disabilities and death, with an enormous health burden, especially among the most vulnerable and poor populations. While P. falciparum is responsible for most deaths, P. vivax is the most widespread of all of the Plasmodium species, can cause severe, even fatal infections and results in significant global morbidity and mortality (Menkin-Smith and Winders, 2020). Recent studies have reported the virulence of P. vivax (Lampah et al., 2011;Tanwar et al., 2011); although, the proportion of cases due to P. vivax reduced from about 7% of the global malaria cases in 2000 to 3% in 2019 (WHO, 2020a).
Malaria is an endemic disease in the tropical and subtropical regions of the world. An estimated 229 million new cases and 409,000 deaths were recorded in 2019 (WHO, 2020a). About 94% of the global malaria cases and 95% of the deaths were recorded in the African region in 2019 (Figure 1), with children under the age of 5 years and pregnant women being the most susceptible groups (WHO, 2020a). Nigeria was responsible for 27% of the malaria cases and 23% of deaths globally in 2019 (WHO, 2020a); this high prevalence illustrated in Figure 2 (WHO, 2018a). This is because Nigeria is the most populous nation in Africa, with P. falciparum being the main cause of malaria (Adebayo and Krettli, 2011;WHO, 2020a). Actually, P. falciparum is the most predominant species in every country in sub-Saharan Africa; the countries with the highest number of malaria cases in West, East, Central and Southern Africa are shown in Table 1. In some countries in Africa, a hundred percent of the population is at risk of the disease (both low and high), such as Nigeria, Mozambique,  Table 1). However, the numbers of reported cases in such countries are low compared to the expected numbers because of lack of access to diagnosis/health facilities or presumptuous treatment ( Table 1). Ethiopia has been reported to have the highest number of P. vivax infections in Africa (WHO, 2020a). The higher prevalence of the disease based on the reported cases in the other regions of Africa compared to Southern Africa (Table 1) is an indication of favorable environmental conditions such as higher temperature and rainfall in such regions (Greenwood et al., 2008).
Drug chemotherapy has a strong historical link to the use of traditional plant infusions in various cultures. Research based on such knowledge has yielded the two most important drugs critical to malaria: the alkaloid quinine present in Cinchona species of highland rain forests in South America and artemisinin from Artemisia annua, first registered in the millenary Chinese medicine.
Artemisinin-based combination therapies (ACTs) are the most recommended treatments for uncomplicated P. falciparum malaria, while artesunate is considered the most effective antimalarial drug for severe cases (Roussel et al., 2017), with several biochemical processes reported as targets in the parasites (Wicht et al., 2020). Despite the safety and efficacies proven for the use of these antimalarial drugs, the emergence of multidrug resistance has unfortunately limited their effects and challenged the field (Nieves et al., 2016). The resistance to ACTs is already spreading from Southeast Asia as reported in 2008 (Noedl et al., 2008), giving rise to a danger alert to other high-poverty regions in the world. The identified artemisinin resistance phenotype is associated with mutation of a kelch domain protein gene (k13), postulated to be involved in the parasite protein trafficking organelles during the intraerythrocytic cycle (Ariey et al., 2014). This mutation has recently been reported in East Africa (Uwimana et al., 2020;Asua et al., 2021), Papua New Guinea (Miotto et al., 2020), and French Guiana (Mathieu et al., 2020); yet, there is limited evidence of delayed parasite clearance after ACT treatment in these regions (Yeka et al., 2016).
The treatment failure associated with ACTs in Africa has been linked to resistance of parasite to the partner drugs and not to artemisinin resistance (Slater et al., 2016). However, Ala675Val mutation in the kelch 13 propeller gene was seen in 5% of isolates in Northern Uganda and this mutation has been known to cause delayed parasite clearance in Southeast Asia (Asua et al., 2019). In the African continent, four countries have identified mutations associated with therapeutic failure of artemisinin derivatives. However, Pfk13 mutations (M476I, P553L, R561H, P574L,

Quinine
Historical records show that in 1638, the countess of Chinchón, wife of the Spanish viceroy in Peru, suffered a severe fever, which was later known as malaria. When ingesting a portion made by the Indians called "quina-quina" the fever subsided, and the continuity of treatment left her cured (Willcox et al., 2004;Ruiz-Irastorza and Khamashta, 2008;de Oliveira and Szczerbowski, 2009). There is also a reference that the use of Cinchona species in medical practice came to Belgium in 1643, when a public health official in Ghent recommended a powder for the treatment of tertian fevers (Jarcho and Torti, 1993). However, the first indisputable reference is the Schedula Romana, a hand bill issued by the Pharmacy of the Collegio Romano in 1649 and again in 1651, containing precise instructions on its dosage and administration (Jaramillo-Arango, 1949). Before 1820, the bark of a tree native to South America named Cinchona, was dried, ground to a fine powder, and mixed into a liquid (commonly wine) before being used (Achan et al., 2011). The Jesuit priests of the Spanish mission took the tree bark to Europe to sell its powder as medicine against intermittent fevers. Later on, two French chemists, Pelletier and Caventou (1820), isolated the active principle by precipitation and crystallization and discovered that the base febrifuge compounds were the alkaloids named cinchonine and quinine. From it, the alkaloids quinidine and cinchonidine were also described (Willcox et al., 2004;Kaufman and Ruveda, 2005). The four alkaloids from Cinchona bark were all effective against malaria, but quinidine and quinine possessed equal febrifugal activity, while cinchonidine was slightly less efficacious. An additional 25 alkaloids related to quinine had been isolated by 1884 and six more were isolated between 1884 and 1941 (Turner and Woodward, 1998). By the turn of the 18th and 19th centuries, Cinchona bark and quinine became widely accepted and used instead of the powdered bark as the reference treatment for intermittent fevers throughout the world (Gachelin et al., 2017).
During the second World War, there was an interruption in the supply of Cinchona bark, which contained a high amount of quinine, from the Java island where the seeds were largely cultivated (Howard, 1931). In the mid-1800s, the structure of quinine ( Figure 3) was elucidated (Kaufman and Ruveda, 2005). Quinine remained the mainstay of malaria treatment until the 1920s, when more effective synthetic antimalarial drugs became available (AdenAbdi et al., 1995). However, the extraction of quinine from the bark of Cinchona does not yield as much as the extraction from the entire tree. The commercial achievement of this bark, almost led to the extinction of the Amazonian trees, and therefore, there was a great need to synthesize the active compounds (Smith and Willliams, 2008).
Quinine is an alkaloid that presents rapid schizonticidal action against intra-erythrocytic malaria parasites; it is also gametocytocidal for Plasmodium vivax and Plasmodium malariae, though there are conflicting reports on its gametocytocidal effect on Plasmodium falciparum (Chutmongkonkul et al., 1992;Tanaka and Williamson, 2011). Quinine has also an analgesic effect, but not antipyretic properties, and prevents the hemozoin crystals from growing by intercalating the quinolone rings between the aromatic groups of the ferriprotoporphyrine molecules (Pelletier and Caventou, 1820;Institute of Medicine, 2004;Weissbuch and Leiserowitz, 2008;Gachelin et al., 2017). For centuries, quinine has been used to treat malaria, and it remains the drug of choice in the treatment of severe malaria; it has been chosen as a second line treatment in combination with antibiotics, after artemisinin combination therapy (ACT) in the treatment of complicated malaria (WHO, 2015).

Resistance to Quinine
Parasite drug resistance is probably the greatest problem faced by malaria control programs worldwide and is an important public health concern. Over the years, malaria parasites have developed resistance to several commonly used antimalarial drugs (Achan et al., 2011). Diminished sensitivity of P. falciparum to quinine has been widely documented in Asia (Sucharit et al., 1979;Bjorkman and Phillips-Howard, 1990;Mayxay et al., 2007), South America (Neiva, 1987;Best Plummer and Pinto Pereira, 2008;Legrand et al., 2008), and Africa (Pradines et al., 1998;Zalis et al., 1998;Mutanda, 1999). However, the development of resistance to quinine has been slow; although its use started in the 17th century, resistance to quinine was first reported in 1910 (Peters, 1982), and after so many years of its discovery, quinine remains an important and effective treatment for malaria today, despite sporadic reports of resistance (Institute of Medicine, 2004). Thus, with increasing resistance in almost all areas with P. falciparum malaria to synthetic antimalarials by the 1980s, quinine again played a key role, particularly in the treatment of severe malaria (AdenAbdi et al., 1995).
Quinine is still used as monotherapy to treat malaria today in most countries of Africa and in some Central American, Caribbean, Eastern Mediterranean, South-East Asian and Western Pacific countries (WHO, 2019). In several settings, the use of quinine for uncomplicated malaria cases has reduced due to toxicity, poor compliance, and the implementation of newer and better tolerated therapies. However, from frequent stock-outs of the recommended ACT and the increasing resistance to chloroquine and antifolates, quinine use in recent times has increased (Wasunna et al., 2008;UMSP, 2010).
The 2015 World Health Organization (WHO) guidelines recommend a combination of quinine and clindamycin, or only quinine to treat pregnant women with uncomplicated P. falciparum and who have chloroquine-resistant P. vivax malaria during the first trimester. For the treatment of severe malaria of adults and children, it is essential that full doses of effective parenteral (or rectal) antimalarial treatment be given promptly in the initial treatment (WHO, 2015). The Cinchona alkaloids quinine and quinidine are the second choice, after artemisinin derivatives. When a complete oral treatment of severe malaria is not possible, but injections are available, the intramuscular quinine could be used if artesunate or artemether is not available (WHO, 2015).

Artemisinin
Qinghao ("blue-green herb") is the Chinese name for a relatively common herbal plant otherwise known as Artemisia annua or sweet wormwood (Maude et al., 2010). Artemisia annua is native to Asia (China, Japan, Korea, Vietnam, Myanmar, Northern India, Southern Siberia and throughout eastern Europe) (WHO, 1998), but it also grows in other countries such as Congo, India, Brazil, Australia, Argentina, Bulgaria, France, Hungary, Italy, Spain, and the USA (Willcox et al., 2003;Zhou et al., 2014). There is an integral interaction between plants, the environment and the production of secondary metabolites, which can directly interfere with their quantities and qualities (Kutchan, 2001). The artemisinin content is affected by several factors, such as geographic conditions, harvest time, temperature and fertilizer application; harvesting the plant at the right time is extremely important to ensure the optimal artemisinin content in A. annua. Thus, for the cultivation of this medicinal plant, it is important to plan the crop establishment for the beginning of the rainy season, and the soil needs to be plowed to a fine slope and consolidated by rolling when appropriate, because the sowing depth is also critical for A. annua (Jelodar et al., 2014). This means that not all A. annua plants have high concentrations of antimalarial compounds.
The A. annua has been used as a remedy by Chinese herbalists for the crafting of aromatic wreaths, as a source of essential oils used in flavoring vermouth, and for more than 2,000 years in Chinese traditional medicine, as a treatment for fever and hemorrhoids (Van Agtmael et al., 1999;Klayman, 1985). Artemisia annua is also used as a source of artemisinin (Zhou et al., 2014), a potent antimalarial drug discovered by the Chinese scientists led by Youyou Tu, that was awarded the 2011 Lasker Prize and the Nobel Prize in Physiology or Medicine in 2015 (Youyou, 2015). The project leading to the discovery of artemisinin was initiated in response to a request from North Vietnamese leaders who were suffering heavy losses of soldiers due to malaria during the Vietnam War (Cui and Su, 2009). The drug is present in the leaves and flowers of the plant accounting for approximately 0.01-0.8% of dry weight (Van Agtmael et al., 1999). Chemically, artemisinin is a sesquiterpene trioxane lactone containing a peroxide bridge (Figure 4), which is essential for its activity (China Cooperative Research Group, 1982).
The earliest known record of A. annua was in the book 52 Prescriptions, discovered in the Mawangdui tomb of the Han Dynasty in 168 BC, where it was first described for the treatment of hemorrhoids (Qinghaosu Antimalarial Coordinating Research Group, 1979;Klayman, 1985). Around 340 AD, in Hong Ge's Handbook of Prescriptions for Emergency Treatment, a cold extraction method of qinghao was described for the treatment of intermittent fevers (Klayman, 1985). Qinghao has also been mentioned in several later standard Chinese Materia Medica texts as treatment for fever (Qinghaosu Antimalaria Coordinating Research Group, 1979;Hien and White, 1993); it has also been used for haemorrhoids, lice, wounds, boils, sores, and convulsions (Hsu, 2006).
The active ingredient of qinghao, artemisinin, was isolated from the plant A. annua in 1972 by a group of Chinese scientists (Hien and White, 1993). In 1975, its unique chemical structure was elucidated, as a sesquiterpene lactone bearing a peroxy group, which is believed to be responsible for the antimalarial activity of A. annua (Willcox et al., 2003), and is quite different from those of all known antimalarial drugs (Liu et al., 1979), then the name qinghaosu ("active principle of qinghao") was changed to artemisinin (Klayman, 1985). Its structure was determined by X-ray analysis in 1979 (Balint, 2001;Krishna et al., 2008). Further studies led to the development of more stable derivatives, such as dihydroartemisinin, artemether, artemotil (arteether) and artesunate (Ansari et al., 2013).
Artemisinin and its derivatives produce rapid parasite clearance, killing young circulating parasites more than the other forms of the parasite, before they are sequestered in the deep microvasculature (Dondorp and Von Seidlein, 2017). This phenomenon is important in the pathogenesis of malaria because mature parasites are able to adhere to endothelial cells, blood cells and platelets, which prevent their circulation in the bloodstream and they will therefore be able to escape clearance by the spleen (Buffet et al., 2011). This is clinically relevant, particularly in the cases of cerebral and severe malaria (Hsu, 2006). These drugs interact with heme to produce carbon-centered free radicals that alkylate protein and damage the microorganelles and membranes of the parasites (Meshnick, 1998), significantly faster than any other antimalarial does (Willcox et al., 2003;Sriram et al., 2004). Unlike the quinine-related drugs and antifolate drugs, artemisinin and its derivatives are also gametocytocidal and reduce the transmission of malaria by P. falciparum (Price et al., 1996).
Since 1994, artemisinins have been used in artemisinin-based combination therapies (ACTs) to treat uncomplicated malaria (Ouji et al., 2018), and by 2006, ACTs had become the recommended treatments for falciparum malaria worldwide (WHO, 2015). The artemisinin derivatives are usually combined with a more slowly eliminated drug in ACTs (Ansari et al., 2013). In this case, the role of the artemisinin compound is to reduce the number of parasites during the first 3 days of treatment (reduction of parasite biomass), while the role of the partner drug is to eliminate the remaining parasites leading to a malaria cure. ACTs are also recommended by WHO for chloroquine-resistant P. vivax malaria, and the injectable artesunate and artemether, are recommended for the treatment of severe malaria (WHO, 2018b).
ACTs are the most effective antimalarial medicines available today, but there are already cases of resistance to artemisinin and its derivatives. Artemisinin resistance in Cambodia was first identified in clinical studies in 2006, and few years later in Myanmar, Thailand, Viet Nam, and Lao. However, retrospective analysis of molecular markers indicated that artemisinin resistance probably emerged in 2001, before the widespread deployment of ACTs in Cambodia (Noedl et al., 2008;Dondorp et al., 2009;WHO, 2017). In spite of the fact that WHO continues to monitor cases of resistance to artemisinin and its derivatives, new drugs must be discovered to overcome the problem of the development of resistance that can make its use unfeasible in the future.

Current Malaria Treatment
ACTs are recommended by WHO as the first-and second-line treatment for uncomplicated P. falciparum malaria, as well as for chloroquine-resistant P. vivax malaria. The role of the artemisinin compound is to reduce the number of parasites during the first 3 days of treatment (reduction of parasite biomass), while the role of the partner drug is to eliminate the remaining parasites, leading to cure (WHO, 2018b). It is recommended to use a 3-day treatment regimen with one of the five different ACTs, namely artemether/lumefantrine, artesunate/amodiaquine, artesunate/mefloquine, dihydroartemisinin/piperaquine or artesunate/sulfadoxinepyrimethamine. ACTs are not recommended in the first trimester of pregnancy for uncomplicated P. falciparum malaria; the World Health Organization guidelines (WHO, 2015) recommends a combination of quinine and clindamycin, or only quinine for uncomplicated malaria caused by P. falciparum and by chloroquine-resistant P. vivax.
In areas with chloroquine-sensitive P. vivax, P. ovale, P. malariae or P. knowlesi infections, the adults and children are treated with ACTs or chloroquine. Primaquine can be administered to avoid relapses of vivax malaria in people with no Glucose-6-phosphate dehydrogenase deficiency. The treatment of adults and children with severe malaria is done with intravenous or intramuscular artesunate for at least 24 h, but if parenteral artesunate is not available, intramuscular artemether is used in preference to quinine (WHO, 2015).

Study Selection and Analysis
The flowchart ( Figure 5) demonstrates the strategy used for identification, and the criteria for inclusion and exclusion of the plants focused in the present review. A total of 321 articles were initially retrieved from the database Pubmed in the last 10 years (2011-2020) using the keywords "antimalarials," "plants" and "natural products." Some additional references of other articles published before have been also included, although they were not found after using the keywords. In a further analysis, articles which focused on the plant species tested in vivo against malaria, using mice infected with Plasmodium species, were selected. This gave a total of 91 full articles, from which 19 articles were excluded because: 1) they were in other languages rather than English; 2) the reported chemosuppression was below 30%, which did not correspond to the criteria for antimalarial activity, as defined below; 3) the tests were with polyherbal extracts rather than with one species of plant; 4) the active plants were toxic; 5) the tests against early sporogonic stages were conducted ex vivo; or, 6) the studies were performed using plants associated or in combination with antimalarial drugs. After applying all these requirements, the total number of publications evaluated was 72, as shown in Table 2 and Figure 5.

RESULTS AND DISCUSSION
The scientific names of the plant species tested, their botanic families, parts of the plants fractionated, the ethnomedicinal uses and the countries where the studies were performed are shown in Table 2. Among the 72 articles, 54 of them evaluated the antimalarial activities of one plant species each while 18 of them evaluated two or three plant species each, giving rise to a total of 90 plant species, some of which were studied by more than one author. The three most studied families were Fabaceae, Apiaceae and Lamiaceae. The plant species evaluated for antimalarial activities corresponded to 44 botanical families; the parts of the plants tested were aerial parts, leaves, leaf latex, rhizomes, roots, root bark, stem bark, whole stem, branches, twigs, petiole, cortex, flower, fruits, fruit pulp, unripe fruits, and whole plant ( Table 2).
A total of 60 of the 90 medicinal plant species (66.67%) tested in mice with rodent Plasmodium species were recommended for the treatment of malaria and/or malaria prevention, as well as for other ailments; six species (6.67%) were used against fever in general, which happens to be the main acute symptom of human malaria. A total of 15 species (16.67%) had an ethnopharmacological recommendation for other diseases and morbidities rather than malaria, while nine species (10%) did not have their ethnobotanical uses mentioned ( Figure 6A).
Further information about the plant species studied are depicted in Table 2. Most studies regarding the antimalarial activities of plant species using animal models, were performed presumably where the plant species occur and/or are native: 59 of the species were in the African continent (65.56%), 18 species were in Asia (20%) and 13 species were in South America (14.44%) ( Figure 6B).
Regarding the countries where the studies were performed in mice infected with malaria parasites, an African country had the highest number, that is Ethiopia (Africa, n 29), followed by Brazil (South America, n 11), India (Asia, n 10) and Nigeria (Africa, n 10) (Figure 7).  Other details of the in vivo tests described in the original articles are summarized in Table 3. As expected, most medicinal plants exhibited antimalarial activities against Plasmodium species in mice experimentally infected (P. berghei or P. yoelli), though some active doses were too high. These doses are not feasible under clinical settings, making such extracts of no clinical relevance. The results validated the ethnobotanical uses of several plants in malaria endemic countries as they reduced parasitaemia and were well tolerated by the uninfected mice ( Table 3).
The active doses of the extracts from medicinal plants tested in mice infected with rodent malaria species ranged from 1 to 6,000 mg/kg, although there was no clear relationship between doses and percentage inhibition of parasite growth, in several cases. The mean survival time was assessed for 65 (72.2%) of the species evaluated; for 55 of them (84.6%), there was an increase in the survival time of the treated mice while for 10 plant species (15.4%) there was no change in the time of animal survival, as compared to the untreated malaria controls ( Table 3). The acute toxicity was evaluated for 40 of the 90 plant species; the LD 50 values for 37 species (41.1%) were 2,000 mg/kg body weight or above (Table 3), and six of the plant species had LD 50 values equal to or higher than 5,000 mg/kg body weight, suggesting that these plant extracts were not toxic (Lorke, 1983). The most active species was Heliantus annuus (popularly know as sunflower) in tests using ethanolic extract of the leaves, which caused a high suppression of parasitemia (49, 76, and 82% using oral doses of 1, 10 and 100 mg/kg, respectively). Its mechanism of action was through the in vitro inhibition of heme polarization by the parasites in a dose dependent manner. Extracts of other parts of the plant species, including roots, stems and flowers (Table 3) were also highly active (Ekasari et al., 2019;Ekasari et al., 2020), but the toxicity of the extracts was not evaluated, perhaps because the sunflower oil from seeds is largely used all over the world in food preparation.
Medicinal plants are often used in health care delivery as complementary and/or alternative medicines against human malaria, specially in poorer areas of the African countries. Such use may be important, especially in conjunction with other health measures like mosquito bed-nets, repellents, and/ or treatments with an antimalarial, including the inexpensive chloroquine pills, that costs about 10 US cents per course of treatment for an adult (Institute of Medicine, 2004).

Entandrophragma cylindricum
Ethyl acetate stem bark extract 99% (250)  with the present analysed data, it is difficult to evaluate the real pharmacological usefulness of medicinal plant extracted in organic solvents to serve as future bioproduct for the development of antimalarials.

Tests of Medicinal Plants From Brazil Against Experimental Rodent Malaria
In Brazil, plants and other natural products are rarely used to treat or prevent malaria. This, in part, is due to the fact that the Public Health system provides diagnosis and treatment of the disease free of charge. The specific diagnosis is promptly provided by the public health system of the Ministry of Health in Brazil, followed by treatment with antimalarials. In addition, human malaria is of compulsory notification to the Public Health system, that also controls the antimalarial drug distribution, under the State governments in Brazil (Ministério, 2020). Treatment is only provided after the parasite species has been parasitologically confirmed, and it varies according to the diagnosis of the species of parasite, which at present is mainly P. vivax followed by P. falciparum. The malaria endemic areas are restricted to the Amazon region in Brazil and its neighbouring countries in South America: Colombia, Peru and Venezuela and the Guyanas (Figure 8).
In the Amazon region, the Quilombolas are endowed with extensive experience in the use of medicinal plants, as they have centuries of close contact with and dependence on local biodiversity as a means of livelihood and therapeutic resources. This fact makes the traditional communities attractive for groups conducting ethno-directed studies for medicinal plants used against malaria and other diseases . The popular uses of one species of  Frontiers in Pharmacology | www.frontiersin.org September 2021 | Volume 12 | Article 734263 medicinal plant, Ampelozyzyphus amazonicus, known as "Indian beer," has been previously described, and found to be frequent among the indigenous people in the State of Amazon (Botelho et al., 1981;Brandão et al., 1985;Carvalho et al., 1991;Brandão et al., 1992;Krettli et al., 2001;Oliveira et al., 2011), and also common among the Quilombolas (Oliveira et al., 2011. It has been demonstrated that the ethanolic extracts of A. amazonicus target the sporozoites, the infective form inoculated through the mosquito bite; in vitro and in vivo models were used in the study (Andrade-Neto et al., 2008). The animals treated with ethanolic extracts by oral route, prior to sporozoite inoculation intravenously, had a significantly prolonged malaria pre-patent period (which is the time relapsed between sporozoite inoculation and detection of parasitemia in the experimental animals). The treated mice had a lower parasitemia and a prolonged survival time, as compared to the untreated control mice infected with sporozoites. Additional in vitro tests clearly confirmed the in vivo results: sporozoites pre-incubated with the plant extracts, were less infective to host cells, as compared to control sporozoites in culture medium. In conclusion, the "Indian beer" ethanolic extract was shown to be a potent prophylactic against malaria (Andrade-Neto et al., 2008). In other studies, it was clearly shown that the extracts of "Indian beer" were inactive against the blood forms of the parasite in mice (Botelho et al., 1981;Brandão et al., 1992).
Unfortunately, very few groups studying the experimental activity of medicinal plants in the world have facilities to produce sporozoites for the in vivo and in vitro tests. Thus, compounds active against these forms, or against the intrahepatic parasites developed from sporozoites will not be discovered until such tests are more easily performed with medicinal plants aiming at new antimalarials. Our group has developed an experimental model to test extracts from plants against the sporogonic stages in mosquitoes as well as the tissue cycle of sporozoite development, using an avian malaria parasite P. gallinaceum and mosquitoes Aedes, which are susceptible to the species (Carvalho et al., 1992).
Another medicinal plant species frequently used to treat fevers and malaria in Brazil is Bidens pilosa, which is active against malaria and was listed as an antimalarial medicinal plant of interest to the Unified Health System in Brazil, largely based on experimental studies of our group Oliveira et al., 2004;Palhares et al., 2015). The ethanolic crude extracts from B. pilosa caused 60% reduction in parasitaemia at doses of 250 mg/kg in mice infected with P. berghei; importantly, the ethanolic extracts from B. pilosa were similarly active in vitro against P. falciparum chloroquine-resistant (clone W2) and chloroquine-sensitive parasites (clone D6) .
Essential oils (EOs) have been considered as an important class of antimalarial natural products with low molecular weight components, rich in monoterpenes and sesquiterpenes, found specially in plants native to Northeast Brazil (Mota et al., 2012). Thus, EOs obtained from Vanillosmopsis arborea (Asteraceae) were active sub-cutaneously, causing up to 47% inhibition of parasitemia in mice. EOs present in Lippia sidoides Cham. (Verbenaceae) and in Croton zehntneri (Euphorbiaceae) were active by oral route, causing 43-55% malaria growth inhibition, respectively, although no dose response activity was observed. The active EOs had monoterpene and phenylpropanoid compounds like estragole, α-bisabolol and thymol active in vitro against P. falciparum but not tested in mice with malaria (Mota et al., 2012). Another plant that contains an oleoresin rich in sesquiterpenes and diterpenes, but with high in vivo activity, is Copaifera reticulata, a tree distributed throughout the Amazon region, which has β-caryophyllene as its major compound, and caused a reduction of 93% in parasitemia when it was tested in mice (de Souza et al., 2017).

Natural Products Isolated From Medicinal Plants and Their Activities
Of all the antimalarial medicinal plants studied from 2011 to 2020, very few have their active principles isolated and characterized, such as Coptis japonica, Heinsia crinata, Piper peltatum and Murraya koenigii. Some of the active principles have been previously isolated from other plants while others are novel. Most of the studies carried out on the active principles are mainly in vitro studies. This is partly due to the fact that these compounds were isolated in small amounts that allowed only in vitro studies, not a main focus of this review. To overcome such bottle-neck, it is important to start the process of isolation of compounds using large amount of plant materials. The isolated compounds evaluated for in vivo antimalarial activities were: 1) Coptisine Chloride from Coptis japonica, (Teklemichael et al., 2020); 2) 4-Nerolidylcatechol isolated from Piper peltatum (Rocha e Silva et al., 2011); and 3) Myristic Acid and β-Caryophyllene from Murraya koenigii (Kamaraj et al., 2014) ( Tables 2, 3).
In the studies with Piper peltatum, very high doses of 4-Nerolidylcatechol, the active principle (400 and 600 mg/kg body weight), have been tested, resulting in chemosuppression of 34.4 and 63.1%, respectively, in P. berghei NK65-infected mice (Rocha e Silva et al., 2011). Such high doses are not clinically feasible for human use; thus, efforts should be directed towards the development of compounds with higher activities at low doses as antimalarial drugs.
The 4-day suppressive test (Peters, 1965) was used for all the in vivo studies. However, one of the studies failed to determine the parasitaemia of day 4 post-inoculation, which is very important for identifying the fast acting compounds against malaria. It is only the slow acting compounds that are left till days 5, 6, and 7 post-inoculation (Fidock et al., 2004).
One of the compounds, Coptisine Chloride, was administered intraperitoneally, which is not the conventional route of administration for in vivo studies in murine malaria. The pharmacokinetic parameters of the drugs administered via such route are different from those of conventionally used routes of administration and therefore, they are not applicable to humans (Teklemichael et al., 2020).
Some pure compounds such as Aspidoscarpine, Uleine, Apparicine, and N-Methyl-Tetrahydrolivacine were isolated from A. olivaceum (Chierrito et al., 2014), Isositrikine, 10-Methoxygeissoschizol and Ramiflorine were isolated from A. ramiflorum , and the bisindole alkaloid Leucoridine B was isolated from A. pyrifolium (Ceravolo et al., 2018); but none of these pure compounds was tested in the experimental mouse model. Braznitidumine was isolated from A. nitidum (Coutinho et al., 2013), but it was not active against P. berghei in experimentally infected mice.
Only one compound was tested against the life cycle in the mosquito, namely, Daucovirgolide G extracted from the plant Daucus virgatus; it is impressive that it inhibits 92% of the early sporogonic stages of the parasite at 50 µg/ml (Sirignano et al., 2019; Table 4). This transmission blocking activity appears to be related to the presence of an intact endocyclic double bond system of the compound, that interacts with the biological target (Sirignano et al., 2019). This structural part is obviously lacking in Daucovirgolide J, which is the reason for its lack of activity (Sirignano et al., 2019).
Africa has a rich flora, ranging from the Savannah to the rainforest, with diversity of phytochemicals and other biomolecules which possess various pharmacological activities, some of which have been extensively reviewed elsewhere (Moyo et al., 2015;Van Wyk, 2015;Ahmed et al., 2018;Oguntibeju, 2018;Van Vuuren and Frank, 2020). Many plants have been reported to be used for the treatment of malaria in the African continent (Soh and Benoit-Vical, 2007;Pillay et al., 2008;Adebayo and Krettli, 2011;Maranz, 2012;Chinsembu, 2015;Memvanga et al., 2015;Omara et al., 2020). As the use of Cinchona species (from which quinine was isolated) for the treatment of malaria dates back to the 1700s (Guerra, 1977;Lee, 2002), the use of some plants, such as Azadiractha indica and Alstonia broonei, for the treatment of malaria, indigenously, in the African continent dates back to centuries, though this has not been properly documented. More recently, the efficacies of some of these medicinal plants have been scientifically authenticated and their active principles isolated, with the mechanisms of action of some of them delineated. However, none of the isolated compounds has been developed into drugs to be used clinically for the treatment of malaria, as reviewed below.
The mechanisms of action of most isolated compounds from the medicinal plants are unclear and/or were only studied in vitro against P. falciparum. This is the case of Coptisine, isolated from Coptis japonica, which exhibits its activity by inhibiting P. falciparum dihydroorotate dehydrogenase, an enzyme required for the synthesis of pyrimidine in the parasite (Lang et al., 2018). The authors reported that the kinetic parameters obtained revealed that coptisine was an uncompetitive inhibitor of the enzyme. Berberine, isolated from Coptis japonica, has been shown to inhibit telomerase activity in P. falciparum (Sriwilaijareon et al., 2002), but there are no documents of studies with their activities in the malaria infected mice.
The chemical structure and molecular formula of each isolated compound are shown in Table 5.

Acute and Sub-Chronic Toxicity of Some Isolated Compounds
Some of the compounds isolated from Coptis japonica (Teklemichael et al., 2020), Piper peltatum (Rocha e Silva et al., 2011) and Murraya koenigii (Kamaraj et al., 2014) plants have been evaluated for their acute toxicities ( Table 4). None of them could be declared safe, having oral LD 50 values that are less than 5,000 mg/kg body weight (Lorke, 1983). Among the compounds evaluated for acute toxicity, Palmatine was the least toxic with the highest oral LD 50 value. However, many of the isolated compounds have not been evaluated for their acute toxicity. This may also be due to fact that these compounds were isolated in small amounts which were not sufficient enough to allow their being tested for acute toxicity. Other compounds were tested by intraperitoneal route   seem to be related to the extracts that have been used against malaria; furthermore, only Coptisine Chloride was tested in vivo (Teklemichael et al., 2020) (Table 3). Other studies have shown that administration of Palmatine, Coptisine and Berberine, at the dose of 156 mg/kg body weight, did not exert nephrotoxic or hepatotoxic effects, though Palmatine significantly increased (p < 0.05) plasma total bilirubin concentration compared to control (Yi et al., 2013). However, the authors did not proceed further to identify whether hemolysis, impaired conjugation of bilirubin in the liver or excretion of conjugated bilirubin into the bile duct was responsible for such increase. Many of the compounds have not been evaluated for sub-chronic toxicity or for chronic toxicity. This has limited the determination of the no adverse effect levels (NOAEL) and low adverse effect levels (LOAEL) of the compounds.

Genotoxicity
Berberine, a compound isolated from Coptis japonica ( Table 4), has been reported not to be genotoxic for prokaryotic cells (Pasqual et al., 1993); however, it has been reported to be genotoxic to dividing eukaryotic cells by intercalating with the DNA (Jennings and Ridler, 1983). Palmatine has been reported to exert genotoxicity causing DNA double strand breaks, though to a lesser extent than Berberine (Chen et al., 2013). Both compounds have been reported to inhibit topoisomerase I and II activities, thereby inhibiting DNA relaxation and decatenation during replication (Chen et al., 2013). Inhibition of DNA decatenation results in the stabilization of topo II-DNA complexes , thereby enhancing the induction of DNA double strand breaks. Palmatine has been reported to interact with DNA by binding to the groove of DNA (Mi et al., 2015). Knipholone and knipholone anthrone have been reported not to cause DNA damage on their own but knipholone anthrone caused DNA damage in the presence of copper ions through the generation of hydrogen peroxide (Habtemariam and Dagne, 2009). 4-Nerolidylcatechol has been shown to exhibit low genotoxicity compared to negative control. However, 4-Nerolidylcatechol has been reported to protect against cyclophosphamide-induced DNA damage by scavenging the free radicals generated by cyclophosphamide (Valadares et al., 2007).

Mutagenicity
It has been reported that berberine, in the absence of microsomal fraction S9, was weakly mutagenic in Salmonella typhimurium TA98, a frameshift detecting strain (Nozaka et al., 1990). It did not cause an increase in the frequency of point mutation (Pasqual et al., 1993) under conditions in which the frameshift mutation was increased by a factor of 4 in Saccharomyces cerevisiae using this model (Pasqual et al., 1993). None of the studies evaluated the mutagenic activities of the compounds using the murine malaria parasite model which analyses the micronucleated cells (Tometsko et al., 1993).

Carcinogenicity, Reproductive Toxicity and Allergenicity
Several purified compounds exert carcinogenic effects after accumulation, over time, in cellular systems. The searches made on PubMed and PubChem revealed that the above isolated compounds have not been evaluated for carcinogenic and allergenic effects. Their effects on pregnancy, such as absorption of fetus, and teratogenic effects have not been evaluated. This indicates that all the isolated compounds have not fully gone through the process of drug development. Consequently, not a single one of them has been developed into an antimalarial drug, neither been subjected to clinical trials.

CONCLUSION AND FUTURE PERSPECTIVES
Of the seventy one manuscripts published in the past 10 years that evaluated the antimalarial activities of extracts and isolated compounds from plant species in rodent malaria model, most of them aimed the erythrocytic stages of the parasite, which are responsible for the malaria symptoms. Only one compound was tested against the early sporogonic stages of the parasite. In addition, a few active principles were described. All steps of extractions, from the pre-extraction to final extraction are equally important in the study of medicinal plants, and play a critical role in the outcomes, including the product yield and phytochemical characterization; and these steps seem to interfere in the final product activity (Azwanida, 2015). Most of the experimental studies of plant materials used organic solvents, in spite of the recommendation in the specific literature that only water and ethanol should be used in traditional preparations for medical use . Taken together, these facts explain the reduced contribution of Africa, in the past 10 years, to the achievement of the Medicine for Malaria Venture objective, which is the development of a new antimalarial drug every 5 years. The reason for this is not far-fetched but in part, it is due to the gross lack of standard facilities for antimalarial drug development in various countries of the African continent, which is responsible for most of the antimalarial tests herein described, especially in countries like Ethiopia and Nigeria, where most of the results have been published in the last 10 years. It is believed that technological advancement in the African continent and the world at large, will certainly go a long way in tackling this fundamental problem, thereby liberating the continent from the scourge of this disease.

AUTHOR CONTRIBUTIONS
IC designed the figures and prepared the tables; AA performed the bibliographic survey of the articles; JA wrote about plant isolated compounds, their toxicity, genotoxicity, mutagenicity and carcinogenicity; AK supervised the work and critically reviewed the manuscript. All the authors contributed to writing and analysing the paper.

FUNDING
This work was supported by a grant from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) 409751/2018-9, and FASESP 2019/19708-0, Brazil. AK is a recipient of a Senior Fellowship from CNPQ, Brazil.